key: cord-0005013-fam92577 authors: Otsuki, K.; Maeda, J.; Yamamoto, H.; Tsubokura, M. title: Studies on avian infectious bronchitis virus (IBV): III. Interferon induction by and sensitivity to interferon of IBV date: 1979 journal: Arch Virol DOI: 10.1007/bf01317496 sha: ef9e5d3478a25dc6971c9013a57eebff7747ac19 doc_id: 5013 cord_uid: fam92577 The induction of interferon by avian infectious bronchitis virus (IBV) and the sensitivity of IBV to interferon were studied. The results of experiments with ten IBV strains are summarized as follows. 1. All the IBV strains tested induced interferon in chick embryo (CE) cells, chicken kidney (CK) cells and embryonated eggs. The Iowa-609 strain induced about 1000 units of interferon in CE cells while the Beaudette-42 strain induced about 200 units of interferon in embryonated eggs; the interferon titers induced by other strains usually ranged from 5 to 60 units. No IBV strain induced interferon in HeLa or BHK-21 cells. 2. IBV particles inactivated by ultraviolet irradiation or by heating lost their ability to induce interferon. 3. The properties of the interferon produced in the present study are similar to those of other interferons produced in chicken cells. 4. HeLa or BHK-21 cells did not acquire resistance to virus infection, after incubation with interferon produced in CE cells. On the other hand, CK cells acquired the same degree of resistance to virus infection as CE cells after incubation with interferon produced in CE cells. 5. All the IBV strains tested were sensitive to interferon in CK cells. The sensitivities of Massachusetts-41 and Holte strains to interferon were similar to that of vesicular stomatitis virus. In 1949, G~OtTP~ (5) reported the production of an interfering material in the allantoie fluid of avian infectious bronchitis virus (IBV)infected embryonated eggs. Later, GRouP~ and PuGH (6) observed that this material interfered with the growth of both influenza virus and IBV in eggs. Then, LOMBA~])I (9) described a similar interference in embryonated eggs produced by the Massachusetts and Connecticut strains of IBV. Furthermore, it has been reported that IBV interferes with Newcastle disease virus (NDV) infection in in vivo (7) , in ovo (14) , and in 0304-8608/79/0060/0249/$ 01.40 K. O~rsl~KI, J. MAEDA, H. YAMAMO~O, and M. TSUBOKURA: vitro (1) systems. It has been shown that these interfering phenomena were not due to the action of interferon. On the other hand, interferon induced by IBV and other coronaviruses has been poorly characterized. 0nly three reports of interferon induction by IBV have been published. Yu~ov (18) observed that the IBV-41 strain induced interferon in ovo, while LOMI~ICZI (11) reported that only the Beaudette-222 strain could induce interferon in chickens, embryonated eggs and chick embryo (CE) ceils. However, GOMEZ and RAGGI (4) failed to induce interferon in vitro or in tracheal organ cultures with the Massachusetts strain. The sensitivities of IBV and other coronaviruses to interferon has not been studied. In this report, ten IBV strains (12, 13) were examined for their ability to induce interferon and for their sensitivities to interferon. Viruses IBV Beaudette-42 (Be-42), Massachusetts-41 (IB-41), Connecticut A-5968 (A-5968), Connaught, Holte, Iowa-609, KI-I, Nerima, Ishida and Shiga strains (12, 13) were studied. Vesicular Stomatitis Virus (VSV) The VSV New Jersey serotype was supplied by the National Institute of Animal Health, Tokyo (Director Dr. S. Shibata). This virus had previously been passaged at least three times in embryonated eggs. Before use, the virus was subcultured twice ill CE cells. Cultured Cells Chicken kidney (CK), CE, I-IeLa and BHK-21 cells were used (12, 13) . Each strain of IBV, at a titer of 10G.5--107-5 TCIDso, was inoculated onto monolayers of CE, CK, geLa or BI{K-21 cells. After adsorption of virus for 90 minutes at 37 ° C, the cells were washed three times with maintenance medium and then cultured at 37 ° C. Culture media were usually collected 24 or 48 hours after inoculation. The media were heated for 15 minutes at 60 ° C to inactivate the virus particles and then centrifuged at 30,000 rpm for 60 minutes to pellet the virus. The supernatant fractions were titrated for interferon. Interferon induction in embryonated eggs was examined as follows. Each strain of IBV at titers of I06.°--I0 v.° TCIDs0 was inoculated into the allantoie cavity of 10-day-old embryonated eggs. Allantoic fluids were collected 48 to 120 hours after inoculation from the infected eggs and analysed as described for the cultured ceils. In most experiments, the interferon titer was determined by placing 3 ml of serial twofold dilutions of interferon onto CE-cell monolayers seeded in small bottles. When an interferon titration was done in CK, I-IeLa or BHK-21 cells, the homologous cells were used to titrate the interferon. The cell monolayers were treated with interferon for 16 hours at 37 ° C, washed three times with Hanks' balanced salt solution, and then infected with VSV at a titer of 50--150 plaque-forming units (PFU). After virus adsorption for 60 minutes at 37 ° C, 5 ml of Eagle's minimum essential medium containing i per cent Baetoagar (DIFCO), 0.i per cent tryptose phosphate broth (DIFCO) and 0.004 per cent neutral red was added, and cultivation was continued for 36 hours at 37 ° C. The interferon titer was calculated as the highest dilution producing a 50 per cent reduction in the number of VSV plaques (PR~0 units). Ultraviolet ( U V ) Irradiation o/IBV 1.5 ml aliquots of culture medium in petri dishes of 6.5 cm diameter were irradiated with a UV lamp (15 W, 0.5 A) at a distance of 7.5 era. The IBV strains were examined for interferon induction in cultured cells. As shown in Table 1 , all the strains except Connaught induced interferon in CE and CK cells. An especially high interferon titer was produced by Iowa-609 strain in CE cells. The other strains induced up to 90 units of interferon in both cell types. All the IBV strains failed to induce interferon in HeLa and BHK-21 cells. The production of interferon in CE cells inoculated with Iowa-609 was examined. Interferon was first observed 6 hours after inoculation (Fig. 1) . The amount of interferon increased gradually and reached a maximum level after 24 hours. Progeny viruses appeared after 6 to 12 hours and the peak virus titer was observed after 36 hours. K. OTSUKI, J. MAEDA, H. YAMA~IOTO, and M. TSUBOKUI~A: The results obtained are shown in Table 2 . The IB-41, Nerima and Ishida strains failed to induce interferon. However, the other seven strains induced up to 190 units of interferon in embryonated eggs. The Connaught strain, which did not induce interferon in CE or CK cells, induced it in eggs. Experiments were done to see whether IBV, inactivated by UV irradiation for 20 seconds or heating for 15 minutes at 60 ° C, induced interferon. 106.0 to 107.0 TCIDs0 of virus was used. All the strains completely lost their infeetivities on irradiation or heating. Furthermore, none of the inactivated strains, inoculated onto CE cells or into the allantoie cavities of 10-day-old eggs, induced interferon within 96 hours of inoculation. Two interferon preparations were used. One was produced in CE cells inoculated with Iowa-609 and had a titer of 1000 units. The other was produced in embryonated eggs inoculated with Be-42 and had a titer of 150 units. These interferons did not sediment on centrifugation at 100,000 × g for 3 hours, were sensitive to treatment with 0.1 per cent trypsin for 1 hour at 37 ° C, and their activities were reduced by 90 per cent by heating for 10 minutes at 100 ° C. Treatment of the interferons at pH 2.0 or pH 10.0 for 24 hours at 4 ° C did not affect their titers. Furthermore, their titers were not reduced by freeze-tha~dng 10 times ; or UV irradiation for 60 seconds. The interferon tested was prepared from CE cells infected with Iowa-609. 250 units of interferon were incubated with HeLa, BHK-21 and CK cells for 16 hours at 37 ° C, and then about 120 PFU of VSV were inoculated onto the cells. Control cells were not treated with interferon, but were inoculated with 120 PFU of VSV. As shown in Table 3 , in interferon-treated HeLa and BHK-21 cells plaque formation by VSV was inhibited slightly, compared with the control, but VSV plaque formation in CK cells was completely inhibited. Interferon produced in embryonated eggs infected with the Be-42 strain was used in this study. The sensitivity of the IBV strains to interferon was assayed in CK cells. Approximately 100 PFU of the IBV strains and VSV were added to interferon-treated and control cultures. Plaque reduction between 20 and 80 per cent was plotted, and the PRs0 units of interferon against each virus was determined. The results obtained are shown in Figure 2 . The IB-41 and Holte strains were as sensitive to interferon as VSV, while the sensitivities of the Be-42, Iowa-609, KH, Ishida and Shiga strains were 30-40 per cent that of VSV. The A-5968, Connaught and Nerima strains were only about one-tenth as sensitive as VSV. As all the IBV strains tested induced interferon in CE and CK cells, and in embryonated eggs we suggest that IBV, like many other tl,NA viruses (8), can induce interferon. However, the ability of IBV to induce interferon appears rather weak, as the interferon titer with most strains was not high and a few investigators (4, t0) have failed to induce interferon with the Massachusetts strain. Furthermore, a large inoculum was required for interferon induction. No interferon was induced in HeLa or BHK-21 cells by any IBV strain, even though the Be-42 and Holte strains grew in BHK-21 cells (13) . Thus, we suggest that IBV can only induce interferon in chicken cells or organs. However, other mammalian cells must be tested to confirm this suggestion. All the IBV strains tested, when inactivated by UV irradiation or heating, lost their ability to induce inteferon. These results agree with those reported previously by Lo~IczI (11) and suggest that only infectious IBV particles can induce interferon. It is not known why inactivated IBV particles cannot induce interferon. Similar amounts of interferon were produced in CK and CE cells on infection with IBV, and furthermore CK cells acquired the same degree of resistance to virus infection as CE cells, when they were incubated with interferon produced in CE cells or embryonated eggs. Thus CK cells can probably be used to test the sensitivity of IBV to interferon. It is clear that IBV, at least in CK cells, is sensitive to interferon. However, unlike some viruses (2, 3, 10, 15, 16) , there is no close relationship between the ability of any strain to induce interferon and its sensitivity to interferon. Similarly, the resistance of the IBV strains to various chemical and physical treatments (12) or their growth in various cells (13) probably bears no relationship to their ability to induce interferon or their sensitivity to interferon. We conclude that IBV is an interferon sensitive virus rather than an interferon inducing virus. However, the interferon sensitivity of the IBV strains should be examined in in vivo systems, as the sensitivity of viruses to interferon is not always the same in different systems (17) . Infectious bronchitis virus interference with Newcastle disease virus in monolayers of chicken kidney cells Growth characteristics, interferon production and plaque formation with different lines of Edmonston measles virus Interferon production and sensitivity of Semliki Forest virus variants Local immunity to avian infectious bronchitis in tracheal organ culture Demonstration of an interference phenomenon associated with infectious bronchitis virus (IBV) of chickens, g Interference between influenza virus and infectious bronchitis virus of chickens, g Separation of Newcastle disease and infectious bronchitis viruses in mixed infections Studio nelto cmbrione di pollo fenomeno di interferenza frata ripe Massachusetts e Connecticut de] virus della bronchite infettiva Studies on interferon production and interferon sensitivity of different strains of Newcastle disease virus Interferon induction by different strains of infectious bronchitis virus Studies on avian infectious bronchitis virus (IBV). I. Resistance of IBV to chemical and physical treatments Studies on avian infectious bronchitis virus (IBV). II. Propagation of IBV in several cultured cells Infectious bronchitis virus interference with growth of Newcastle disease virus. I. Study of interference in chicken embryos Relationship between viremia and interferon production of Japanese encephalitis virus Interferon production by different viruses Relative sensitivities of viruses to different species of interferon Interferon formation in chick embryos infected with one or two viruses Authors' address: Dr. K. OTS~I, Department of Veterinary Microbiology, Faculty of Agriculture, Tottori University, Tottori City, Japan.Received August 15, 1978